KR20160029868A - Oscillating amplification reaction for nucleic acids - Google Patents

Abstract

One embodiment of the present invention is to provide a method for amplifying a template of a target chromogenic sequence contained in a sample. The method comprises contacting the sample with an amplification reaction mixture containing a primer complementary to the template of the target nucleic acid sequence. The reaction temperature was several times During the temperature cycle, the difference between the maximum and minimum temperatures is about 20 ° C. The template of the target nucleic acid sequence is amplified.

Description

Round-trip amplification reaction of nucleic acids {OSCILLATING AMPLIFICATION REACTION FOR NUCLEIC ACIDS}

Cross-reference to related applications

[0001] This application claims the benefit and priority for the application of US Provisional Patent Application No. 61/477,437 filed on April 20, 2011 under the title of "Oscillating Amplification Reaction for Nucleic Acids" And the specification and claims are incorporated herein by reference.

[0002] This application is in the application of US Provisional Patent Application No. 61/477,357 filed on April 20, 2011 under the title of "Integrated Device for Nucleic Acid Detection and Identification" Claims for interest and priority, the specification and claims are incorporated herein by reference.

Reference to research or development that has been aided by the federal government

[0003] No application

Compact Consolidation by reference of substances submitted on disk

[0004] No application

Copyright material

[0005] No application

Sequence listing, table, or reference to a computer program

[0006] Applicants submit here as a sequence listing the text submitted under the heading 041812_ST25.txt of 10K kilobytes generated on April 20, 2012, which is ASCII compliant and incorporated herein by reference.

Field to which the invention belongs (technical field):

[0007] Embodiments of the present invention provide a method for template-dependent amplification of a target nucleic acid sequence by reciprocating the reaction temperature within a narrow range, preferably no greater than 20°C, in any given thermal polymerization cycle, and It relates to the device.

[0008] The discussion below refers to a number of publications and references. Here, such discussion is intended to give more complete background knowledge on scientific principles, and should not be construed as acknowledging that such publications are prior art for the purpose of determining patentability.

[0009] Nucleic acid amplification is one of the most indispensable techniques in molecular biology widely used in research, genetic testing, agriculture and forensics. The most common amplification technique is polymerase chain reaction (PCR), in which the distribution of a specific nucleic acid target sequence in solution increases exponentially (see US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). The PCR reaction employs two oligonucleotides that hybridize to opposite strands of the DNA double helix upstream (5') or downstream (3') of the target sequence to be amplified. DNA polymerase (generally thermostable) is hybridized in the 5'->3' direction by adding deoxynucleoside-triphosphate (dNTPs) to copy the target sequence and generate a double-threaded DNA product. It is used to extend the old primer. By cycling the temperature of the reaction mixture (typically 95°C), the two strands of DNA are subsequently at a high temperature to serve as a template for the binding and polymerization of the primers at lower temperatures (e.g. 55°C and 60°C). Separated. After repeating this process several times, a single target sequence can be amplified into billions of copies.

* [0010] Although PCR is a gold standard amplification method in a well-equipped laboratory, expensive and complex thermal cycling and skill with active heating and cooling heating elements and accurate temperature controllers in order to obtain meaningful results It is a bit complicated, requiring an experienced technician. For example, most PCR reactions require rapid and accurate cycling between at least two temperatures (e.g. 95 degrees and 57 degrees Celsius), which is typically an expensive and energy-inefficient Peltier engine (thermal electric cooling mechanism) and Leads to the use of correct temperature control elements. These inherent limitations make PCR incompatible with the development of useful and cost-effective field diagnostic nucleic acid diagnostics in the absence of an experimental infrastructure to support them. In an effort to eliminate some of the strong financial requirements of PCR, various'isothermal' amplification techniques have been developed over the past decades. In such a reaction, the nucleic acid is amplified at a single temperature, eliminating the need for an expensive thermal cycle and making it easier to use in low cost diagnostic devices. Examples include nucleic acid sequence-based amplification (NASBA), helicase mediated amplification, strand replacement amplification, and loop-mediated isothermal amplification (LAMP). However, these isothermal amplification techniques often require an amplification time of 60-90 minutes (due to the slow in vitro dynamic enzymatic procedure) and precise temperature control at a single temperature point in order to acclimate to the extremely stringent amplification reactions, which are used for field diagnosis. It again causes the lack of robustness and speed required for the application.

[0011] Template-dependent nucleic acid amplification is the cornerstone of nucleic acid-based molecular diagnostics. Robust, low-cost, rapid on-site diagnostics nucleic acid diagnostics are urgently needed in health care, agriculture, and in the context of biological terrorism and war. However, conventional analytical chemistry strategies involving PCR or isothermal amplification have made such amplification approaches expensive and impractical for financing locations where nucleic acid-based molecules can make the greatest impact on emerging disease prevention and control. It imposes engineering and robustness limits. For locations where there is no dedicated laboratory infrastructure, significant improvements still have to be made in nucleic acid amplification that will provide adequate and robust diagnosis.

[0012] Traditional PCR relies on highly specific and rapid thermal cycling, changing the temperature as often as 40°C. In addition to maintaining the solution temperature accurately, such amplification methods require expensive equipment for rapid heating and (especially) cooling of the PCR reaction mixture. While isothermal nucleic acid amplification procedures eliminate the need for complex thermal cycling mechanisms, they are generally slow (at least 60 minutes reaction time), not reliable, and require accurate temperature measurements.

In the PCR thermal cycling process, the PCR cycler must have good temperature control to maintain the temperature in the sample, and typically at least 2° C. per second heating (and/or cooling) rate. Temperature control is typically achieved by a feedback loop system, whereas temperature uniformity is achieved by a highly thermally conductive but bulky material such as copper. High heating rates are obtained by performing a proportional integrated derivative (PID) control method, which is limited by the maximum permissible loss and heat capacity. High cooling rates are somewhat difficult to achieve, and bulky systems have thermoelectric elements (P. Wilding, MA Shoffner and LJ Kricka, Clin. Chem., 1994, 40, 1815-1817.) (often called Peltier elements) or Other means such as water (JB Findlay, SM Atwood, L. Bergmeyer, J. Chemelli, K. Christy, T. Cummins, W. Donish, T. Ekeze, J. Falvo, D. Patterson, J. Puskas, J. Quenin, J. Shah, D. Sharkey, JWH Sutherland, R. Sutton, H. Warren and J. Wellman, Clin. Chem., 1993, 39, 9, 1927-1933). These PCR machines are complex and power-intensive devices. Because the systems are bulky, their thermal time constants are in minutes rather than seconds, which leads to long transition times and unwanted PCR byproducts. The high power consumption makes it impossible to make a battery-operated and transportable PCR system.

[0014] Recently, with the development of micromachining and biological micro-electromechanical systems (bioMEMS) based on silicon technology, many groups around the world have begun to develop micro PCR (μPCR), which is a lab-on-a-chip or micro-total analysis. It is the heart of the system (μTAS). The researchers follow two basic approaches: a stationary system with circulating temperature and a flow system with three zones at different temperatures. Both systems have their own advantages and disadvantages. The stop system cycles the temperature of the chamber to change the temperature of the PCR solution. They don't need a pumping system or other means to get the PCR sample down. Flow-through systems typically have three constant temperature zones. Only the sample changes the temperature by moving between zones of different temperatures. This type of PCR system is faster than the first one, but requires the implementation of a mechanism to move the sample. In both cases, the heater is integrated with the PCR system, so emptying the device is not economical to avoid cross contamination after only one test run. The main advantage described by these two formats is the reduced cycle time by using a smaller sample volume compared to traditional devices. However, these PCR chips use a substrate material such as silicon, which requires the adoption of expensive and complex fabrication procedures resulting in very high unit costs. In addition, as a result of the reaction volume (<μL), which is extremely small to obtain an increased surface-to-volume ratio, and the type of material used in the μPCR chip, conventional methods including non-specific absorption, inhibition, sample evaporation, and foam formation of biological samples. In PCR Some effects, which are not very common, become noticeable. Another current effort is to develop a temperature cycling reaction microchip that integrates a stop chamber and continuous flow PCR, giving fluidity to changes in the number of cycles and temperature zones within the stop chamber PCR chip, while the flow-through microchannel PCR chip. It is related to performing an effective temperature cycling However, the efficiency of the hybrid PCR device is still being confirmed, and the issues related to sample inhibition, absorption and foaming associated with the μPCR approach are all prior sample preparation/nucleic acid separation processes and amplification reagents, such as extremely high polymerase concentrations, Remarkable stringency remains imposed on reaction conditions such as PCR primer concentration.

One embodiment of the present invention is to provide a method of amplifying a template of a target nucleic acid sequence contained in a sample. The method includes contacting a sample with an amplification reaction mixture containing a primer complementary to a template of the target nucleic acid sequence. The reaction temperature is reciprocated between the upper and lower limit temperatures with a temperature difference of up to about 20° C. during several temperature cycles. The template of the target nucleic acid sequence is amplified.

One embodiment of the present invention provides that the temperature difference is up to about 15° C. during several temperature cycles. Another embodiment provides that the temperature difference is up to about 10° C. over several temperature cycles. Another embodiment provides that the temperature difference is up to about 5° C. over several temperature cycles. The temperature may be varied by (+/-2° C.) for a given temperature and/or range according to an embodiment of the present invention.

Another embodiment of the present invention provides that, after reaching the upper or lower limit temperature, the temperature is maintained for a predetermined time within the temperature fluctuation. Or, after reaching the upper limit temperature or the lower limit temperature, the temperature is changed to another temperature within the temperature range. In one embodiment, the lower limit temperature is at least about 50°C. In other embodiments, the upper temperature limit is at most about 85°C. The upper and lower limit temperatures may vary by about +/- 5°C according to one embodiment.

According to one embodiment of the present invention, the template of the target nucleic acid sequence may be single-stranded DNA or RNA, double-stranded DNA or RNA, RNA, DNA, or a combination thereof. The length of the target nucleic acid may be shorter than 1000 bp, and may be shorter than 250 bp, shorter than 150 bp, or shorter than 100 bp.

[0018] One or more embodiments may include a pair of primers that bind to opposite strands of the nucleic acid template. The primer pair may have a length and GC content such that the melting point is 65°C. In another embodiment, the primer pair is one having a length and GC content such that the melting point is 70°C. For example, each primer of a primer pair independently has a length of 35 to 70 base pairs. According to an embodiment of the present invention, the melting point of each primer of the primer pair is 70 to 80°C. In a preferred embodiment, the primer pair comprises a forward primer and a reverse primer each having a length of 40 to 47 base pairs.

[0019] Yet another embodiment of the present invention provides a method for amplifying the template of the target nucleic acid sequence contained in the sample. The method includes contacting a sample with a primer or an amplification reaction mixture comprising a primer or a pair of primers having a length of 35 to 70 base pairs and complementary to the template of the target nucleic acid sequence, and the melting point of each primer of the primer pair is 70 to 80°C. Includes. The amplification reaction mixture also contains DMSO, monovalent cations, divalent cations, dNTPs and DNA polymerase. The reaction temperature is reciprocated between the upper and lower limit temperatures with a temperature difference of up to about 20° C. during several temperature cycles, and the template of the target nucleic acid sequence is amplified. In a preferred embodiment, the divalent cation is selected from the group consisting of magnesium, manganese, copper, zinc, or combinations thereof, and the monovalent cation is sodium, potassium, lithium, rubidium, cesium, ammonium, or a combination thereof. It is selected from the group. In another preferred embodiment, the amplification reaction mixture contains a nucleic acid destabilizing agent. In a more preferred embodiment, the amplification reaction contains a DNA polymerase, which may be a heat stable DNA polymerase. The DNA polymerase may be selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase, but is not limited thereto, and other polymerases disclosed herein or known in the art may also be included. The DNA polymerase may comprise strand replacement activity. In another embodiment, the DNA polymerase does not have 3'->5' exonuclease activity. In another embodiment, the method of amplifying the template further comprises a reverse transcriptase and a DNA polymerase. For example, reverse transcriptase is a heat stable reverse transcriptase. The reverse transcriptase may be selected from AMV-RT, Superscript II reverse transcriptase, Superscript III reverse transcriptase, or MMLV-RT, but other reverse transcriptases known in the art may also be used, and the present invention is not limited thereto. Another embodiment of the invention further comprises adding the single stranded binding protein to the reaction mixture as disclosed. For example, a single-stranded binding protein is a heat-stable single-stranded binding protein or a heat-stable single-stranded binding protein.

[0020] Yet another embodiment of the present invention provides a mixture containing single-stranded or double-stranded nucleic acid unstable agents. For example, there is dimethyl sulfoxide (DMSO) or formamide, but other agents such as glycerol may be added for the same purpose, and are not limited thereto.

[0021] Another embodiment of the present invention provides a method the sample is not an alcohol-free or and or the sample is not a salt-free.

[0022] in another embodiment of the present invention there is provided a method for amplifying a target nucleic acid sequence in a sample containing a template, where the amplification reaction mixture is a single-stranded or double-stranded nucleic acid unstable agents; Monovalent cation; Divalent cations; dNTPs; And DNA polymerase buffered to a pH capable of supporting activity.

[0023] Another embodiment of the invention provides an amplification reaction mixture containing one or more of the following: a single-stranded or double-stranded nucleic acid unstable agents; Monovalent cation; Divalent cations; dNTPs; And DNA polymerase buffered to a pH capable of supporting activity. The DNA polymerase may be a heat stable DNA polymerase. The DNA polymerase may be selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase, but is not limited thereto. DNA polymerases may have strand replacement activity. The DNA polymerase can be selected as having no 3'->5' exonuclease activity. The mixture may also contain one or more of the following: a destabilizing agent which is a single stranded binding protein, dimethylsulfoxide (DMSO) or formamide; A divalent cation that may be a salt selected from the group consisting of magnesium, manganese, copper, zinc, or combinations thereof, and a monovalent cation that is a salt selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium, or combinations thereof.

[0024] Objects, advantages, and additional ranges of novel features and applicability of the present invention will be described in part within the detailed description below, which is considered together with the accompanying drawings, and those skilled in the art based on the following tests It will be partially apparent to, or may be learned by the practice of the invention. The objects and advantages of the invention can be realized and obtained by means of the means and combinations particularly pointed out in the accompanying claims.

[0025] The accompanying drawings, which are incorporated into and form part of this specification, describe one embodiment of the present invention, and together with the detailed description, serve to explain the principles of the present invention. The drawings are only for the purpose of describing preferred embodiments of the invention and are not to be construed as limiting the invention. The drawing is as follows:
1 is a panel A showing a representative trace of thermal fluctuation observed during several cycles of OPCRar (gray line) compared to a traditional two-step PCR reaction (black line), and nucleotide amplification according to an embodiment of the present invention With panel B describing the method, a schematic diagram of a round trip PCR amplification reaction according to an embodiment of the present invention is shown.
Figure 2. Panel AE shows a series of photographs of an acrylamide gel showing another polymerase used to generate a 153 base pair (bp) product according to an embodiment of the present invention.
3. Panel AB shows a series of photographs of an acrylamide gel showing the effect of ethanol on nucleic acid amplification according to an embodiment of the present invention.
4 is a photograph of an acrylamide gel showing the difference between the temperature of annealing and the melting point of the primer (melting temperature) supporting efficient amplification according to an embodiment of the present invention.
5 is a photograph of an acrylamide gel showing the effect of hot-start DNA polymerase on the formation of a primer dimer according to an embodiment of the present invention.
6 is a photograph of an acrylamide gel showing the effect on the primer-dimer formation of the GC and AT clamp according to an embodiment of the present invention.
7 is a photograph of an acrylamide gel showing the effect on product formation of a single-stranded binding protein according to an embodiment of the present invention.
8. Panel AB is a T4 gene protein according to an embodiment of the present invention
Pictures of the acrylamide gel are shown describing the reduction in the amount of primer-dimer formation by 32.
9 is a photograph of an acrylamide gel showing a specific target sequence present in the amplified double-threaded DNA according to an embodiment of the present invention.
[0035] Figure 10 is This is an acrylamide gel photograph describing amplification of a specific target sequence present in ssDNA according to an embodiment of the present invention.
11 is a photograph of an acrylamide gel showing amplification of a specific target sequence present in plasmid DNA according to an embodiment of the present invention.
12 is a photograph of an acrylamide gel showing amplification of single-stranded RNA according to an embodiment of the present invention.
13 is a photograph of an acrylamide gel showing amplification of a specific target sequence in bacterial genomic DNA according to an embodiment of the present invention.
14 is a photograph of an acrylamide gel showing amplification of a specific target sequence present in chloroplast DNA according to an embodiment of the present invention.
15 is It is an acrylamide gel photograph showing amplification of two target sequences according to an embodiment of the present invention.
16. Panel AB shows a picture of an acrylamide gel showing amplification of a target sequence in the presence of SSB at a low melting point according to an embodiment of the present invention.
[0042] Figure 17 panel AB shows a picture of an acrylamide gel showing amplification of the target using precise temperature control and/or rapid ramping parameters as required for a typical PCR thermocycler.
18 is a photograph of an acrylamide gel showing amplification of amplified target Rbcl in a low cost heater without ramping or precise temperature control.

[0044] The term'nucleic acid' as used throughout the specification and claims refers to single-stranded or double-stranded DNA, RNA or DNA/RNA hybrid molecules. Single-stranded nucleic acids can have secondary structures such as hairpins, loops and stem elements. Double-stranded or single-stranded nucleic acids can be linear or circular. Double-stranded nucleic acids can be intact or interstitial. The double-stranded molecule may have a blunt end or a single-stranded protruding end. Nucleic acid samples can be isolated from cells or viruses, and chromosomal DNA generated in cells or viruses, extra-chromosomes including plasmid DNA, recombinant DNA, DNA fragments, messenger RNA, ribosomal RNA, transfer RNA, double-stranded RNA Or other RNAs. Nucleic acid can be any number of sources, such as agriculture, food, environment, fermentation or saliva, blood, nose or lung aspirate, cerebrospinal fluid, sputum, feces, milk, mucosal tissue wipes, mucosal tissue, tissue samples, or biological fluids such as cells. It can also be separated from Nucleic acids may be isolated in vitro, cloned, or synthesized. Within the scope of the nucleic acids described above, individual nucleotides may be subject to chemical alterations or modifications such as methylation. Such transformation or alteration may occur naturally or may occur by in vitro synthesis.

[0045] The term'target nucleic acid'or'template nucleic acid' as used throughout the specification and claims means a single-stranded or double-stranded DNA or RNA fragment or sequence intended to be selectively amplified. The size of the nucleic acid to be amplified is defined by the upstream (5') and downstream (3') boundaries, and may be less than 500 bp, preferably less than 250 bp, more preferably less than 150 bp, and even better. It may be less than 100bp. The target nucleic acid may be a fragment contained within a long double-stranded or single-stranded nucleic acid, and may be all double-stranded or single-stranded nucleic acids.

[0046] The term'duplex ' as used throughout the specification and claims refers to a DNA or RNA nucleic acid molecule that is entirely or partially double-stranded.

[0047] The term'thermal cycle' as used throughout the specification and claims means the repeated temperature fluctuations required for nucleic acid amplification to occur. The thermal cycle may include a high temperature melting or denaturing step and a low temperature annealing or hybridization step, but is not limited thereto.

[0048] The term'melting 'or'denaturing' as used throughout the specification and claims means separating all or part of the two complementary strands of a nucleic acid duplex at high temperature. The melting or denaturing temperature can be influenced by the length and sequence of the oligonucleotide primers, the concentration of duplex unstabilizing reagents such as DMSO and formamide, and the ionic strength or pH of the solution.

[0049] The term'annealing 'or'hybridizing' as used throughout the specification and claims refers to sequence specific binding of an oligonucleotide to a single-stranded nucleic acid template. The sequence can only bind to its complementary sequence on one of the template strands, and not to other regions of the template. The specificity of annealing or hybridization can be influenced by the length and sequence of the oligonucleotide, the temperature at which binding is performed, the concentration of duplex destabilizing reagents such as DMSO and formamide, and the ionic strength or pH of the solution.

[0050] The term'primer ', as used throughout the specification and claims, is a single-stranded nucleic acid or oligo capable of binding to a single-stranded region on a target sequence in a sequence-specific manner, enabling polymerase-dependent replication of the target nucleic acid. Means nucleotide.

[0051] The term'OPCRar' used throughout the specification and claims refers to oscillating PCR amplification reaction, which is less than in a typical amplification technique, a temperature fluctuation between denaturation temperature and annealing temperature, such as It is an in vitro technique for amplifying nucleic acids using temperature fluctuations of less than 20°C, preferably less than 15°C, and more preferably less than 10°C.

[0052] The term'accessory protein' as used throughout the specification and claims refers to any protein capable of stimulating activity, for example a thermostable single-stranded binding protein (SSB), such as rec A or RPA (replica protein A), but is not limited thereto.

In one embodiment of the present invention, there is provided a method for amplifying a specific nucleic acid target exponentially by thermal cycling, wherein the temperature fluctuation is preferably less than 20 °C, more preferably less than 15 °C, Even better, it is 10℃. This is a step of providing a single-stranded template of the nucleic acid to be amplified, an oligonucleotide primer for hybridizing to the nucleic acid template, and using a hybridized oligonucleotide to produce a double-stranded extension product complementary to the template strand by means of a DNA polymerase. And repeating the above steps to exponentially amplify the selected nucleic acid target.

Referring now to Figure 1, there is a schematic diagram of a round trip PCR amplification reaction (OPCRar) according to an embodiment of the present invention is described in Panel A, which is compared with a conventional two-step PCR reaction (black line) , Shows representative traces of thermal fluctuations observed over several cycles of OPCRar (gray line). Note the dramatic reduction in temperature fluctuations in OPCRar. 1B shows that the double-stranded target nucleic acid enters the melting step according to an embodiment of the present invention, where partial or complete denaturation of the duplex may be caused depending on the temperature. The unwinding of the duplex begins at the end of the target, and the single-stranded nucleic acid is bound and stabilized by a single-stranded binding protein (marked by a circle). The reaction is cooled and allowed to enter the hybridization/polymerization step, where the primer hybridizes in a specific manner to the 5'end of each strand of the target duplex. After hybridization of the primers, DNA polymerase (squares) binds to the template/primer duplex, copying the DNA template strands, and extending the primers in the 5'->3' direction by integration of dNTPs. If the polymerase used has strand replacement activity, it will be able to replace the opposite strand in the partially denatured complex. As new duplex DNA is generated, the thermal cycle is repeated several times, resulting in exponential amplification of the target nucleic acid sequence.

[0055] In one embodiment of the present invention, thermal cycling is a temperature that reciprocates or circulates between two temperatures where ΔT is preferably 20° C. or less, more preferably 15° C. or less, and even more preferably less than 10° C. It is related. The higher of the two temperatures may be sufficient to denature the double-stranded target DNA or, preferably, to cause only partial denaturation of the double-stranded DNA target. When a high or low temperature is reached, the temperature is maintained for a defined period of time or preferably immediately changes to another temperature.

In a further embodiment of the present invention, the nucleic acid target may be a double stranded nucleic acid such as double-stranded DNA, or may be a single stranded nucleic acid such as single stranded RNA or DNA. If the target nucleic acid is double-stranded, it must be fully or partially denatured by heat or enzymes to form a single-stranded template or template region necessary for DNA polymerase activity and amplification. The length of the target nucleic acid may be less than 1000 bp, preferably less than 250 bp, more preferably less than 150 bp.

[0057] In a further embodiment of the present invention, the oligonucleotide primer used for amplifying the target nucleic acid is a primer pair that binds to the opposite strand of the specific double strand of the target nucleic acid, wherein one primer is upstream of the 5'end of the target. And one primer binds downstream of the 3'end. Under complex conditions, one or more pairs of oligonucleotide primers can be used to simultaneously amplify multiple nucleic acid targets in the same reaction mixture. Oligonucleotide primers may have a length and GC content such that, under generally accepted PCR buffering conditions, the melting point is greater than 65°C, preferably greater than 70°C.

In addition, in a further embodiment of the present invention, the DNA polymerase used is preferably TAQ DNA polymerase, VentR DNA polymerase, DeepVentR DNA polymerase and similar thermostable DNA It is selected from polymerase. Preferably, the DNA polymerase retains strand replacement activity and does not have 3'->5' exonuclease activity (see Fig. 1B). In addition, if the template nucleic acid is single-stranded RNA, the reverse transcriptase used is AMV-RT, Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), Superscript III reverse transcriptase (Invitrogen) and similar thermostable enzymes. Will be selected from

Other-possible thermalphilic polymerases

[0059] Thermal affinity DNA polymerase

[0060] Polymerase and (vendor)

[0061] VentR ( VentR) ^® (Neve)

[0062] VentR ( VentR) (EXO-) ^® (Neve)

[0063] Deep Vent (Nev)

[0064] Deep VentR (Exo-) (Nev)

[0065] Tag (Nev)

[0066] PyroScript (PyroScript) (Lucigen)

[0067] Pyrophage (PyroPhage) ^® 3173, wild type (Lucigen)

[0068] LongAmp Tag (Neve)

[0069] Bst polymerase

[0070] Fire Hot Start II (Nev)

[0071] Phusion High Fidelty DNA polymerase (Neve)

[0072] Fusion (Phusion) (Nev)

[0073] Fusion Flash (Phusion ^® Flash) (Nev)

[0074] 9 Nm (Nev)

[0075] DyNAzyme II Hot Start (Nev)

[0076] DyNAzyme EXT ( DyNAzyme EXT) (Neve)

[0077] DreamTag (Permentas)

[0078] Tag (Natural) (Permentas)

[0079] Maxima Hot Start Tag (Maxima ^® Hot Start Tag) (Permentas)

[0080] Pfu (recombination), (permentas)

[0081] Bsm (Large Short), (Permentas)

[0082] TrueStart ^® Hot Start Tag (Permentas)

[0083] Tfi (Invitrogen)

[0084] AmpiTag (AmpiTag ^® ) (Invitrogen)

[0085] AmpliTag Gold ^® (Invitrogen)

[0086] Platinum (Platinum ^® ) Pfx

[0087] In a further embodiment of the invention, the reaction mixture preferably comprises a single stranded binding protein (SSB) such as a heat stable SSB or a T4 gene 32 protein isolated and cloned from a thermophilic organism.

[0088] In addition, the enzyme preparation is preferably in a single- or double-stranded nucleic acid destabilizing agents such as dimethyl sulfoxide (DMSO) or formamide, are contained in a concentration of 8-15% of the total reaction volume. Alternatively, other agents such as glycerol deaza-dGTP, 3dazopurein, and dITP may be used alone, or may be used in combination with the agents listed above.

[0089] Embodiments of the present invention are ideally suited for use in low cost in situ nucleic acid diagnostics, where a microfluidic bed is placed over a heating element. By reducing the temperature range cycling requirements, a relatively simple heating with a passive cooling mechanism can be used to rapidly cycle the temperature of the reaction solution. The lowered maximum temperature during thermal cycling minimizes fluid evaporation, which can negatively affect the amplification reaction as a whole. More importantly, the robustness of the amplification is greatly improved compared to the conventional PCR procedure, which allows the new method to accommodate temperature fluctuations (inaccurate temperature control) during the amplification process. The specific reaction chemistry of the present invention operates over a wide range of melting (e.g. 70-105° C., not particularly susceptible to foaming) and hybridization temperatures, eliminating the need for a uniform temperature over the entire reaction volume. Appeared to be. Finally, an embodiment of the present invention is a method of separating the nucleic acid at hand by eliminating the alcohol-based washing (e.g. alcohol or isopanol) and centrifugation, heat-drying or vacuum steps between the nucleic acid extraction steps associated with traditional nucleic acid extraction chemistry. It performs well in the presence of alcohols and salts (e.g. -10% ethanol), greatly reducing stringency.

[0090] An embodiment of the present invention includes detection of a pathogen in a biological sample in which the pathogen nucleic acid is a target nucleic acid. Alternatively, the present invention may be used to detect differences in chloroplast DNA, where a fragment of chloroplast DNA becomes a target nucleic acid. In this method, a single base polymorphism may be detected within a target nucleic acid derived from the same or different sources.

[0091] An embodiment of the amplification technique of the present invention is referred to as a'reciprocal PCR amplification reaction' (OPCRar). OPCRar is based on, but is not limited to, a combination of a double-stranded destabilizing agent that lowers the reaction melting point temperature and an oligonucleotide primer having a particularly high melting point (Tm) to increase the annealing temperature during a given thermal cycle. In this method, the in vitro amplification of the target nucleic acid can be performed by rapidly thermal cycling between temperatures of preferably 20°C, more preferably 15°C, and even more preferably 10°C (Fig. 1A). ). Oligonucleotide primers specific for the upstream (5') and downstream (3') regions of the target nucleic acid hybridize to the template, allowing elongation by DNA polymerase to amplify the target. If the DNA polymerase used is a strand replacement polymerase without exonuclease activity, complete thermal denaturation of the double stranded target nucleic acid is unnecessary while working with a duplex destabilizing agent to lower the melting point. The temperature cycling procedure is repeated, resulting in exponential amplification of the specific nucleic acid target sequence (Figure 1B). In OPCRar, the double-stranded target nucleic acid enters a melting step, where depending on the temperature can cause partial or complete denaturation of the duplex. The unwinding of the duplex begins at the end of the target, and the single stranded binding protein (circle) binds to and stabilizes the single stranded nucleic acid. The reaction is cooled and allowed to enter the hybridization/polymerization step, where the primer hybridizes in a specific manner to the 5'end of each strand of the target duplex. After primer hybridization, DNA polymerase (squares) binds to the template/primer duplex and extends the primer in the 5'->3' direction by incorporating dNTPs while copying the template strand of DNA. If the polymerase used has strand replacement activity, it will be possible to replace the opposite strand in the partially denatured complex. With the generation of new duplex DNA, the thermal cycle is repeated several times, resulting in exponential amplification of the target nucleic acid sequence.

[0092] The OPCRar method is based on using a combination of a nucleic acid destabilizing agent that lowers the reaction melting temperature (melting point) and two oligonucleotide primers having an unusually high melting point to increase the annealing temperature during thermal cycling. It is not limited. For a given target nucleic acid, one oligonucleotide primer hybridizes to the 5'-end of the sense strand, preferably comprising the target sequence, and one primer is the antisense strand, preferably comprising the reverse-complementary target sequence. Hybridizes at the 5'-end. OPCRar preferably uses a strand replacement DNA polymerase without exonuclease activity, but is not limited thereto, in order to further lower the melting or denaturation temperature required for efficient amplification of the target nucleic acid. OPCRar can amplify target nucleic acids in the presence or absence of accessory proteins. Any specific OPCRar system can be optimized by addition, deletion or substitution in the mixture.

[0093] This amplification technique has improved properties compared to other amplification techniques reported in the art in terms of low cost, rapid, and on-site nucleic acid diagnosis. Unlike the aforementioned nucleic acid amplification techniques, the OPCRar system made possible by a robust enzymatic procedure is robust, fast, and tolerant of temperature fluctuations in low-cost heating devices, making it ideally suited for low-cost and on-site diagnostic applications. By minimizing the temperature difference encountered during thermal cycling, OPCRar combines the speed and reliability of PCR with the lower equipment requirements of isothermal amplification techniques.

[0094] The simplified thermal cycling requirements of the OPCRar system are ideally suited for passive-cooling appliances, where heat can be applied to one side of the chamber, and cooling takes place by the atmospheric dissipation of heat on the opposite side. Such manual-cooling dramatically lowers the cost and complexity of any nucleic acid diagnostic device. Manual-cooling has previously been reported for use in diagnostic devices, but these devices employ traditional PCR cycling assay chemistry to amplify target nucleic acids while limiting the reaction rate (Luo et. al. Nuc Acids Res. 2005). ; Wilding et. al., Nuc. Acids. Res. 1996; Burke et. al., Science 1998;). Another advantage of OPCRar is that effective nucleic acid amplification can occur over a wide range of melting and annealing temperatures, resulting in less stringent temperature control mechanisms. In the construction of micronized nucleic acid diagnostic structures, maintaining a uniform temperature over the entire reaction volume can be challenging, with particularly high temperature gradients occurring between the heated and unheated sides of the reaction chamber. Such temperature fluctuations can lead to inefficient amplification using conventional PCR or isothermal reaction chemistry. OPCRar is designed to minimize the problems associated with precise temperature control and maintenance, by using a combination of robust polymerase, destabilizing reagents and other polymerase accessory factors: the coolest area of the reaction chamber for a given nucleic acid target. As far as the minimum possible melting temperature and maximum possible annealing temperature are seen, the reaction will proceed effectively even if the other regions of the reaction volume change to >10°C. In addition, the robust OPCRar nature of the amplification chemistry, with minimal power/energy consumption, makes the rigorous sample preparation/nucleic acid separation process at hand (highly pure and at the same time without any traces of contaminants such as salt and alcohol carryover and inhibitory substances). Large volumes (e.g. instead of sub μL in a typical μPCR chip), greatly alleviating the requirements for ultra-high concentration and ultra-purity of PCR enzymes and biologics) and highly concentrated nucleic acids in terms of acquiring sub μL of the inlet template). 20 μL) to enable rapid and effective amplification.

Solvent reagent

[0095] solvents such as DMSO and dimethylformamide are known to be added for each 1% by volume to lower the melting point of duplex nucleic acid ~ 0.5-0.7 ℃. These reagents are often used to improve the amplification efficiency of target nucleic acids having high GC content and consequently high Tm, thereby promoting complete denaturation of double-stranded templates that are difficult to dissolve. Usually the PCR thermal cycling temperature is kept constant by adding a duplex destabilizing reagent to the PCR reaction. On the other hand, OPCRar preferably utilizes the addition of a special high concentration of DMSO to dramatically lower the melting point of the thermal cycle. In traditional PCR, DMSO is rarely used above 10% v/v due to the high temperature of the melting step (typically above 90° C.) and the loss of polymerase activity associated with the high concentration of these reagents. On the other hand, the OPCRar system and method preferably uses a DMSO concentration of 10 to 15%. Unexpectedly, this amount of DMSO does not cause a significant loss of polymerase activity.

Referring now to Figure 2, amplification according to an embodiment of the present invention is compatible with various DNA polymerases depending on conditions. Ribonucleic acid (0.3 ng/μL) isolated from nasal aspirate containing influenza A virus was used as a nucleic acid template under multiple reverse transcription-OPCRar conditions using VentR polymerase. The 153 bp product was generated using OPCRar primers FP3 and RP4, which was visualized by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide. All reactions included Superscript III reverse transcriptase, in which, prior to thermal cycling, the initial cDNA generation step was carried out at 55° C. for 5 minutes. The gel lane was labeled with the concentration of DMSO and the melting and hybridization step temperature used: the reaction was carried out for 40 cycles.

[0097] While conventional PCR enzymes such as Taq DNA polymerase reaction mixtures are extremely sensitive to any trace amount of alcohol such as ethanol, in one embodiment of the present invention, the new reaction mixture is highly resistant to inhibition by ethanol. There is resistance. Referring now to Figure 3, the effect of ethanol on the amplification of nucleic acids according to an embodiment of the present invention is shown. Total nucleic acid (3.4 ng/μL) isolated from leaf tissue infected with Candidatus . Liberibacter asiaticus was used as a starting template. VentR (exo-) DNA polymerase in the presence of 15% DMSO, OPCRar conditions using Et SSB and primers hyvl_For and hyvl_Rev (Fig. 3A) or conventional PCR conditions using Taq polymerase and primers HLBas-P2 and HLBr-P1 The reaction was carried out under (Fig. 3B). The OPCRar solution was heated at 85° C. for 2 minutes to denature the template, and then 40 cycles of reciprocating between 76° C. for 10 seconds and 60° C. for 10 seconds were performed to produce a 139 bp product. A typical PCR reaction was heated to a temperature of up to 95° C. for 2 minutes, followed by 40 cycles varying between 10 seconds at 95° C. and 40 seconds at 58° C. to produce a 130 bp product. The amplified product was visualized on a 12% acrylamide gel stained with ethidium bromide. It shows the ethanol concentration contained in the amplification reaction mixture. It is clear that the OPCRar formulation (Figure 3A) is dramatically more resistant to inactivation by ethanol compared to conventional PCR (Figure 3B).

[0098] Typical conditions under OPCRar VentR (exo -) DNA polymerase and the use of Et SSB was also not cause any loss of the activity by 10% ethanol. This is a very remarkably surprising discovery regarding the application of OPCRar to a low-cost field diagnostic device. Because the common wisdom of PCR and isothermal amplification typically advises users to provide an alcohol- and salt-free, highly purified nucleic acid input. As a result, most of all researchers, prior to putting the sample through the PCR amplification process, between alcohol-based washing and extracting the target nucleic acid from nucleic acid-affinity microbeads, glass frits, matrices or filters, etc. It adopts vacuum drying, air drying, spin down or heating steps. As an example of an integrated point-of-care device, in addition to the amplification and detection of nucleic acids, the device must also rapidly isolate target nucleic acids. Typically this is done by binding the nucleic acids to a glass fiber matrix, washing in the presence of significant concentrations of salts and alcohols, and then extracting them in a buffer containing minimal salts and free of alcohol. Prior to extraction, the wash buffer retained on the binding matrix is removed to prevent carry over to the extraction volume; In commercial nucleic acid separation kits this is typically done by centrifugation. OPCRar's specific enzymatic mixture eliminates the need to carefully remove ethanol during nucleic acid separation, so that embodiments of the present invention provide for low-cost integrated diagnostics that do not require vacuum, centrifugation, air drying or heat drying elements. It makes it possible to be customized.

primer

[0099] Oligonucleotides as described herein can be prepared and purified by methods known in the art (see, eg, US Pat. No. 6,214,587). In this embodiment, two sequence-specific primers representing a primer pair are used to amplify the target nucleic acid sequence exponentially. The first primer hybridizes to the upstream 5'region of the target nucleic acid, and the second primer hybridizes to the downstream 3'region of the target sequence. The primer hybridizes to 5'of one strand present in the target duplex, and the primer is extended by polymerase in the direction of 5'to 3'using the target nucleic acid sequence as a template (Fig. 1B). The conditions of hybridization are standardized as described in the literature ("Molecular Cloning and Laboratory Manual" 2nd edition, Sambrook, Rich and Maniatis, pub. Cold Spring Harbor (2003)). To obtain specific amplification of a given target sequence, homologous primers are preferred, where all nucleotides in the primer are complementary to the target sequence. However, the primer may contain a base that is non-homologous to the template nucleic acid, or may contain a 5′ sequence that is not complementary to the target nucleotide sequence. In order to simultaneously amplify multiple nucleic acid targets in the same reaction mixture, multiple pairs of primers may be used in a single OPCRar experiment. So-called multiplexing is a commonly used technique for single base polymorphism analysis, pathogen detection, and for the integration of internal controls into individual reactions. In addition, a high level of multiplexing can also be achieved through the use of a 5'generic tag sequence introduced into each target specific 3'region, allowing generic amplification of all target sequences with different internal pathogen sequences.

[00100] The design oligonucleotide primers, a melting point and an intra-is related to the number of parameters, such as the primer sequence arrangement-and inter. The melting point is controlled by factors such as primer length and GC content. Inter-primer sequence complementarity can lead to hairpin structure, which can interfere with efficient amplification, while intra-primer homology can produce primer-dimer dubbed unwanted amplification products. When designing a primer, it is important to select a sequence within the target that is specific for the nucleic acid molecule to be amplified and will minimally interact with itself or other primers present during the amplification reaction.

In most nucleic acid amplification strategies, the melting point of the primer is preferably about 10 to 30° C. higher than the temperature at which hybridization and amplification occur. With the temperature of the annealing/polymerization step in the PCR reaction being 55-60° C., the primers are typically 18-30 base pairs in length. In order to allow easy primer binding without loss of sequence specificity, the length of these specific oligonucleotides is minimized. However, in the OPCRar system, the primers are designed to have a different length of 35-55 base pairs and preferably have a melting point of 70-80°C in order to increase the temperature of the annealing step. Considering the level (~10-15%) of the duplex destabilizing agent, DMSO used in a typical OPCRar reaction, it is preferred that the calculated Tm of OPCRar is only <10°C above the annealing temperature used during the thermal cycle. In experiments with the extreme length of the OPCRar primer, efficient amplification occurred despite minimal differences (compensating for the concentration of DMSO) in primer Tm and annealing/elongation temperature.

[00102] Turning now to Figure 4, OPCRar primer by requiring a minimum difference between the annealing temperature and the primer Tm, supports the efficient amplification in accordance with one embodiment of the invention. Primers hyvl_For and hyvl_Rev were used to amplify the plasmid (12 ng/μM) containing the hyvl gene sequence to produce a 139 bp product, which was visualized by electrophoresis on a 12% acrylamide gel stained with ethidium bromide. All reactions were initially subjected to a melting step at 85° C. for 2 minutes, followed by 40 cycles of 10 seconds each at the indicated melting and hybridization temperatures.

The calculated Tm for the primers hyvl_For and hyvl_Rev were 72.2°C and 70.9°C, respectively. The reaction was carried out in 10% DMSO, lowering the Tm by 7° C. assuming a decrease of 0.7° C. per 1% volume. Even with a negligible difference between the primer Tm and the hybridization temperature, it was observed that the amplification reaction proceeds efficiently as if the temperature difference was much smaller.

[00 103] Now looking at Figure 5, for the hot forming primer dimers with OPCRar - shows the effect of the start DNA polymerase. Control reactions containing no nucleic acid template were performed using Superscript III RT, and Taq or platinum Taq DNA polymerase in the presence of primer pairs FP3 and RP4 (8 μM each) and 15% DMSO. Cycling parameters were 40 cycles of 5 minutes at 55°C, 2 minutes at 85°C, and 15 seconds at 80°C and 15 seconds at 65°C. After electrophoresis on 12% acrylamide gel, the OPCRar product was visualized using ethidium bromide. Platinum Taq is a commercially available hot-start enzyme (Invitrogen, Carlsbad, CA), which is conjugated with an antibody that is isolated by heating the reaction solution to 94° C. under conventional PCR conditions. In the presence of the template nucleic acid, this primer pair will produce a 153 bp product. In reducing the formation of <110 bp primer dimers during OPCRar, it will be apparent that this hot-start preparation is not better than the conventional Taq. One problem with long primers used in OPCRar reactions is that they tend to be more prone to producing unwanted non-specific amplification products known as primer dimers. The primer dimer is formed when the 3'end of the primer oligonucleotide is temporarily bonded to each other during the initial temperature increase at the time of the amplification reaction. During this critical period, the DNA polymerase, especially if the concentration of the starting template nucleic acid is very low, can elongate these transient complexes, resulting in products that compete with specific target amplification during thermal cycles. A technique commonly employed to reduce primer dimer formation during PCR is the use of so-called'hot-start' DNA polymerases. These commercially available enzymes are non-covalently bound to inhibitory molecules such as antibodies. When the reaction temperature exceeds 90° C., the inhibitory molecules separate and allow the polymerase to function normally. However, surprisingly, platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), a hot-start enzyme in the present inventors' water, did not significantly reduce the amount of primer dimer amplification, which is a commonly used technique that is sufficient for OPCRar. This indicates that the OPCRar process is a much more effective amplification process than traditional PCR, so as to enable dimer formation resulting from transient dynamic collisions of isotype and heterologous dimers that do not typically occur in conventional PCR procedures.

[00 104] Turning now to Figure 6, in accordance with one embodiment of the invention, a gel showing the effect on the formation of primer dimers of GC and AT are given clamp during OPCRar. In the absence of the starting template, a primer designed to amplify the elongation factor of C. reveribacter asiaticus (3.5 ng/μL) was used to determine the tendency of primer dimer formation. In the presence of the template, the size of the product for the various primer sets will range between 140-155 bp. Primer sequences can be seen on the right with gray AT or GC clamps. For the'mod' primer set, each primer contains a base that is non-homologous to the template (underlined), which increases homology with the second primer. All reactions were carried out in the presence of 15% DMSO, using VentR (exo-) DNA polymerase, Et SSB. The solution was heated at 85° C. for 2 minutes to denature the mold, followed by 30 or 40 cycles of reciprocating between 15 seconds at 80° C. and 15 seconds at 65° C. The amplified product was visualized on a 12% acrylamide gel stained with ethidium bromide. As clearly observed, the GC clamp set caused significant primer dimer formation, while the AT clamp primer did not cause primer dimer formation.

[00105] In order to minimize potential primer dimer formation, OPCRar primers can be designed by employing several strategies different from those used to generate conventional PCR primers. First, PCR primers have a GC-rich 3'end, commonly referred to as a'GC clamp', which results in remarkably specific binding to the target sequence. However, in the OPCRar primer, it was observed that the high GC content in the 3'region of the primer leads to the formation of a larger primer dimer, and thus the OPCRar primer has the affinity of the 3'-3' primer interaction resulting in this undesired amplification product. In order to reduce energetically, the AT is made to include a rich 3'region (FIG. 6). A second strategy for OPCRar primer design is to design a primer comprising a complementary 5'or internal sequence of at least 5 consecutive nucleotides. Oligonucleotides designed in this way will modulate any primer hybridization during an initial increase in reaction temperature towards a duplex structure that is not competitive for polymerase elongation. If an appropriate complementary sequence cannot be found in the target nucleic acid sequence, non-homologous or mutated bases can be used to generate them in the OPCRar primer. The exceptional length of the OPCRar primer overcomes the minimal mismatch between the primer and the target during the initial cycle of the amplification reaction. The primer set is as follows:

EU _ AT

Forward direction

GTTCTTGTAG CGTTGCAGTC TTCTGCGGAA GATAAGGAAT TGCTTT (SEQ ID NO 21)

Reverse

GGGCACGTTT ATTAGCAACA ATAGAAGGAT CAAGCATCTG CACAGAAAT (SEQ ID NO 22)

EU _ GC

Forward direction

CTTGTAGCGT TGCAGTCTTC TGCGGAAGAT AAGGAATTGC TTTCTGCG (SEQ ID NO 23)

Reverse

CACGTTTATT AGCAACAATA GAAGGATCAA GCATCTGCAC AGAAATCACCG (SEQ ID NO 24)

EU - Atmod

Forward direction

GGTGTTCTTG TATCGTTGCA GTCTTCTGCG GAAGATAAGG AATTGCTTT (SEQ ID NO 25)

Reverse

GTAATGGGCA CGTTTATTAG CAACGATAGA AGGATCAAGC AACTGCACAG AAAT (SEQ ID NO 26)

EU _ GCmod

Forward direction

CTTGTATCGT TGCAGTCTTC TGCGGAAGAT AAGGAATTGC TTTCTGCG (SEQ ID NO 27)

Reverse

GGCACGTTTA TTAGCAACGA TAGAAGGATC AAGCATCTGC ACAGAAATCA CCG (SEQ ID NO 28)

[00106] OPCRar primer adenine "A", thymine "T", guanine "G", or a cytosine "C" of the deoxy of ribonucleotide bases, and / or A, C, uracil "U", ribonucleotide bases G It may include any one of one or more. In addition, OPCRar primers may comprise one or more modified deoxy ribonucleotides or ribonucleotide bases, wherein the modifications do not interfere with primer hybridization to the target nucleic acid, prime extension by polymerase, or denaturation of the duplex nucleic acid. OPCRar primers can be modified with a chemical group such as methylphosphonate or phosphorothioate, with a non-nucleotide linker, with biotin, or with a fluorescent label such as an amine-reactive fluorescein ester of carboxyfluorescein. Such modifications can enhance primer performance or facilitate detection and characterization of amplification products.

Polymerase

While [00107] OPCRar, after the primer is hybridized to the single stranded template nucleic acid region, causing a polymerization process. If the target nucleic acid is DNA, a DNA polymerase acting on the target is selected to extend the hybridized primer along the nucleic acid template in the presence of four dNTP nucleotide bases to form a double-stranded product. The strand is complementary to the nucleotide sequence of the template (Figure 1). If the initial target is RNA, reverse transcriptase is first used to copy the RNA template into the cDNA molecule, which is further amplified by DNA polymerase in OPCRar.

[00108] Various DNA polymerases can be selected for OPCRar based on thermal stability and progression, particularly in the presence of unstabilized agents and alcohols (FIG. 2). Although not required, a polymerase that exhibits strand replacement activity and lacks exonuclease activity was found to significantly improve the OPCRar response (FIG. 2 ). Examples of suitable DNA polymerases include Taq polymerase, KlenTaq DNA polymerase (AB Peptides, (St Louis, MO)), Bst DNA polymerase large fragments (New England Biolabs, Beverly, MA), VentR or VentR (exo-). (New England BioLabs), DeepVentR or DeepVentR (exo-) (New England BioLabs) and similar enzymes. Suitable thermostable reverse transcriptases include Superscript II (Invitrogen, Carlsbad, CA), Superscript III (Invitrogen) and similar enzymes. It should be noted that published conventional PCR amplified polymerase mixtures fail to perform OPCRar due to the unique robust requirements of OPCRar amplification. All selected polymerase and raw reagent components should be carefully evaluated and tested experimentally prior to use.

Single stranded binding protein

[00109] The OPCRar system preferably minimizes the temperature difference between the melting and annealing thermal cycle steps, where this temperature difference is the lowest if complete denaturation of the nucleic acid duplex is not essential. While strand-replacement DNA polarmerases are helpful in this regard, accessory proteins can be used to further lower the thermal requirements for efficient amplification. Single-stranded binding proteins (SSBs) are known to stabilize single-stranded nucleic acids to prevent annealing of double-stranded duplex formation, and have been shown to increase the efficiency of nucleic acid amplification reactions. It was found that the addition of thermally stable SSB to the OPCRar method according to the invention results in increased activity (Fig. 7). Although the selection of SSB is not limited to a specific protein, and may include an SSB isolated and cloned from a thermophilic organism or an SSB engineered from a non-thermal stable precursor SSB, for example, Et SSB (BioHelix Corporation, Beverly, MA ).

[00110] Referring now to Figure 7, a gel showing the effect of the single-stranded binding protein on the formation of OPCRar products according to an embodiment of the present invention is presented. A typical influenza A single-stranded DNA template (1E6 copy/μL, Biosearch Technologies, Inc., Novato, CA) was amplified using OPCRar primers FP3 and RP3 to produce a 133 bp product, which was stained with ethidium bromide. % By electrophoresis on an acrylamide gel. OPCRar according to one embodiment of the present invention was carried out in the presence or absence of heat-stable SSB under the following conditions: i ) 15% DMSO; ii ) 15% DMSO, 5% glycerol; iii ) 15% DMSO, 0.25 M betaine. The cycling parameters used for all reactions were 15 seconds at 75°C and 15 seconds at 65°C, and repeated 45 times.

[00111] In order to reduce the formation of primer dimers during the initial heating of the OPCRar solution, in addition to the heat-stable SSBs that help OPCRar, non-heat-stable SSBs such as T4 bacteriophage SSB (New England BioLabs) may be used (FIG. 8). It was observed that the amplification of unwanted primer dimers during OPCRar was minimized by pre-incubating the OPCRar primers in the presence of a molar excess of the T4 gene 32 protein and then adding them to the reaction mixture. These SSBs are hypothesized to bind to single stranded oligonucleotide primers while reducing the potential for 3'-3' coupling and thus formation of primer dimers. Upon heating the solution above 65° C., the T4 SSB denatures, releasing the primer for normal reactivity during the thermal cycle. Referring now to Figure 8, the gel shows a reduction in the amount of primer-dimer formed during one embodiment of the present invention using pre-incubation with the primer and T4 gene 32 protein. Prior to addition of the reaction mixture, OPCRar primers were used in the presence of 1X ThermoPol buffer (New England BioLabs, Beverly, MA) at 25° C. for 5 minutes, the indicated stoichiometry of the active units of the T4 gene 32 protein (T4 SSB). It was cultivated with a theoretical plethora. The synthetic generic flu A DNA template (1E6 copy/μL, Biosearch Technologies, Inc.) was amplified using primers FP3 and RP3 to generate a 133 bp sequence product. The reaction was carried out at 85° C. for 2 minutes, followed by 50 cycles of 15 seconds at 75° C. and 15 seconds at 65° C. The reaction product was visualized by electrophoresis on a 12% acrylamide gel stained with ethidium bromide. The experiment described in FIG. 7 was repeated by another researcher on another day, and the reproducibility of the data was calculated and shown in FIG. 8. It is evident that pre-incubation of the primer with T4 SSB not only increases the amount of amplified product, but also decreases the intensity of the primer dimer band (~100 bp) compared to the case without pre-culture (the first right lane). Was observed.

[00112] The OPCRar technique is a device as described in a generally owned provisional patent application filed on the same day as the subject under the title "Integrated Device for Nucleic Acid Detection and Identification" It is particularly well suited for use in The appearance of a particular embodiment of the device allows the solution temperature to be rapidly cycled while the solution is held in the same chamber, preferably no active cooling. For example, the temperature may be increased or decreased sufficiently to perform OPCRar within 20 seconds or less, more preferably 15 seconds or less, or more preferably 8 seconds or less, or even more preferably 4 seconds or less. I can. Thus, the OPCRar temperature cycle can be performed in a small amount of time, even faster than 8 seconds.

Example One: OPCRar On by DNA Target Duplex Amplification method

[00113] In order to describe that OPCRar has the ability to amplify a specific target sequence present in a double-dead DNA analyte, the present inventors used two OPCRar primers, primers HLB (Huang Long Bing) ForSh and primer HLBRevSh. Using, a 140 bp sequence was generated by the OPCRar system from a PCR-amplified fragment of the C. Liberibacter asiaticus elongation factor gene. OPCRar buffer (10X) was prepared in advance, and 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100 were included. 20 μL of OPCRar solution was prepared by mixing the following:

8.4 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets HLBForSh and HLBRevSh (4 μM each)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single stranded binding protein (500 μg/mL)

2.0 μL PCR product dilution (starting concentration 0.6 to 0.0006 ng/μL)

The reaction was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles were performed while repeating between 3 seconds at 80° C. and 5 seconds at 65° C. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. The 140 bp product was clearly observed from all the dilutions shown, which was consistent with the expected length of the OPCRar target sequence (FIG. 9).

Referring now to Figure 9, the amplification of a specific target sequence present in the double-threaded DNA according to an embodiment of the present invention is shown in the gel. Serial dilutions of PCR-amplified fragments of the C. Liberibacter asiaticus elongation factor gene (0.6 to 0.0006 ng/μL) were used as a starting template. In order to generate the 140 bp sequence, the reaction was carried out using VentR (exo-) DNA polymerase, Et SSB and primers HLBForSh and HLBRevSh in the presence of 15% DMSO. The reaction was heated at 85[deg.] C. for 2 minutes to denature the mold, and then 40 cycles were performed, reciprocating between 5 seconds at 80[deg.] C. and 5 seconds at 65[deg.] C.. The OPCRar product was visualized on a 12% acrylamide gel stained with ethidium bromide. A 140 bp product matching the expected length of the target sequence was clearly observed in all dilutions shown.

Example 2: OPCRar Single stranded by DNA Target amplification method

[00116] To demonstrate that OPCRar can amplify specific target sequences from single-stranded DNA templates, the present inventors used the OPCRar primers FP3 and RP4, which are commercially available universal influenza A template (Biosearch Technologies, Inc.). From the OPCRar system, a 153 bp sequence was generated. 10xOPCRar buffer contains 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. 20 μL of OPCRar solution was prepared by mixing the following:

8.4 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets FP3 and RP4 (8 μM each)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single stranded binding protein (500 μg/mL)

2.0 μL single stranded DNA template (1E9-1E2 copies/μL).

[00 117] as a sensitivity comparison, using the same mold as that used for the concentration of said OPCRar was performed real-time PCR reaction. 10x ThermoPol (New England BioLabs) contains 200 mM Tris-HCl (pH 8.8), 100 mM ammonium sulfate, 100 mM potassium chloride, 20 mM magnesium sulfate and 1% Triton X-100. 15 μL of RT-PCR solution was prepared by combining the following:

9.7 μL water

1.5 μL 10x ThermoPol buffer

0.4 μL dNTPs (10 mM)

Includes 1.5 μL primer set UniAfCDC/UniArCDC (4 μM each), TaqMan probe UniApCDC (1 μM).

0.4 μL Taq polymerase (5 U/μL)

1.5 μL single stranded DNA template (1E9-1E2 copies/μL)

[00118] A series of dilutions of universal influenza A single-stranded DNA template between 1E9 and 1E2 copies/μL were amplified by OPCRar in the presence of 15% DMSO and by real-time PCR using a TaqMan probe. The OPCRar reaction was first heated to 85° C. for 2 minutes, then 80° C. for 15 seconds and 65° C. for 15 seconds, repeated 40 times. The RT-PCR reaction was heated to 95° C. for 2 minutes, followed by 45 cycles between 95° C. 10 seconds and 58° C. 40 seconds. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. A 153 bp product was clearly observed for all samples (FIG. 10), consistent with the expected length of the OPCRar target sequence.

[00119] Referring now to FIG. 10, there is a gel showing the target sequence present in the amplified single-stranded DNA template according to an embodiment of the present invention. Universal Influenza A single stranded DNA template (1E9 to 1E2 copies/μL, Biosearch Technologies, Inc.) was amplified by OPCRar using primers FP3 and RP4 in the presence of 15% DMSO, resulting in a 153 bp product, which was a 12% acrylamide gel stained with ethidium bromide. Images were visualized by electrophoresis (left panel), as a comparison, the same dilution was used as a starting template for real-time PCR using the primer set UniAfCDC/UniArCDC and the TaqMan probe UniApCDC (right panel). The OPCRar reaction was first heated to 85° C. for 2 minutes, then followed by 40 cycles of reciprocating between 80° C. for 15 seconds and 65° C. for 15 seconds. The RT-PCR reaction was heated to 95°C for 2 minutes, followed by 45 cycles of 10 seconds at 95°C and 40 seconds at 58°C. It is clear that OPCRar, when properly optimized, has similar sensitivity to conventional PCR reactions.

Example 3: Method for amplifying specific sequences present on plasmid DNA by OPCRar

[00120] In order to describe that OPCRar can amplify a specific target sequence present in the double-threaded plasmid DNA, the present inventors used two OPCRar primers, primer hyvl_For and primer hyvl_Rev, by the OPCRar system by the OPCRar system. Liberibacter asiaticus hyvl A 139 bp sequence was generated from the plasmid containing the gene. OPCRar buffer (10X) was made in advance and contained 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. By mixing the following, 20 μL of OPCRar solution was prepared:

9.4 μL water

2.0 μL 10x OPCRar buffer

2.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets hyvl_For and hyvl_Rev (8 μM each)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single stranded binding protein (500 μg/mL)

2.0 μL C. Healthy DNA (17.2 ng/μL) extracted from healthy tissues infected with Liberibacter

[00121] Titration with DMSO was performed from 13-8% v/v. The reaction was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating at 80° C. for 10 seconds and 65° C. for 10 seconds were performed. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethynyl bromide. A 139 bp product was clearly observed for all samples (FIG. 11 ), which was consistent with the expected length of the OPCRar target sequence.

Haebomyeon [00 122] With reference now to Figure 11, the gel of the specific target sequence present in the plasmid DNA amplification in accordance with one embodiment of this invention. A plasmid containing the C. Liberibacter asiaticus hyvl gene (17.2 ng/μL) was used as a starting template, and the concentration of DMSO was titrated from 13-8% v/v. All reactions were performed using VentR (exo-) DNA polymerase, Et SSB and primers hyvl_For and hyvl_Rev. The reaction was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 80° C. for 10 seconds and 65° C. for 10 seconds were performed. The OPCRar product was visualized on a 12% acrylamide gel stained with ethidium bromide. A 139 bp product consistent with the expected length of the target sequence was clearly observed at all DMSO concentrations tested.

Example 4: Amplification methods of the human pathogenic virus OPCRar the RNA target sequence present in the aspirate in the nose

[00123] In order to describe that OPCRar can amplify a specific target sequence present in a single-stranded RNA template, the present inventors use the OPCRar primer pair, FP3 and RP4, to be infected with or not infected with influenza A virus. A 153 bp sequence was generated from ribonucleic acid isolated from clinical nasal aspirate. OPCRar buffer (10X) was prepared in advance and contained 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. 20 μL of OPCRar solution was prepared by combining the following:

9.3 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets FP3 and RP4 (8 μM each)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single-stranded binding protein (500 μg/mL)

0.1 μL Superscript III reverse transcriptase (200 U/μL)

2.0 μL 12% nucleic acid isolated from clinical nasal aspirate (0.3 ng/μL)

[00124] The reaction is incubated for 5 minutes at 55° C. for cDNA generation, heated to 85° C. for 2 minutes to denature the template, and then 40 cycles reciprocating between 10 seconds at 80° C. and 10 seconds at 65° C. Performed. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. A 153 bp product was clearly observed in the positive clinical sample, but was not found in the negative clinical sample (FIG. 12 ), which was consistent with the expected length of the OPCRar target sequence.

[00125] Referring now to FIG. 12, there is shown a gel describing a specific target sequence present in single-stranded RNA amplified according to one embodiment of the present invention. Ribonucleic acid (0.3 ng/μL) isolated from aspirate of the nose infected or uninfected with influenza A virus was used as a template. All reactions were performed in the presence of 15% DMSO, using Superscript III reverse transcriptase, VentR(exo-) DNA polymerase, Et SSB and primers FP3 and RP4. The reaction was incubated at 55° C. for 5 minutes for cDNA production, heated to 85° C. for 2 minutes to denature the template, and then 40 cycles were performed, reciprocating between 80° C. for 10 seconds and 65° C. for 10 seconds. The OPCRar product was visualized on a 12% acrylamide gel stained with ethidium bromide. A 153 bp product was clearly observed in the positive clinical sample, consistent with the expected length of the target sequence, but was not observed in the negative sample.

Example 5: Method for amplification of target sequences derived from pathogenic plant bacteria by OPCRar

[00126] To describe that OPCRar can amplify specific target sequences present in the pathogenic bacterial genome, the present inventors used the OPCRar primer pair, EU523377-F-57 and EU523377-R-56, to infected plant tissue From the total nucleic acid isolated from, a 213 bp fragment of the Liberibacter asiaticus elongation factor gene was generated. OPCRar buffer (10X) was prepared in advance and contained 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. 20 μL of OPCRar solution was prepared by combining the following:

8.4 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets EU523377-F-57 and EU523377-R-56 (4 μM each), primer EU523377-F-57 is biotinylated (5') or non-biotinylated

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single-stranded binding protein (500 μg/mL)

2.0 μL Total nucleic acid (1.1 ng/μL) isolated from plant tissue infected with Liberibacter asiaticus

[00127] In order to demonstrate that the primer modification is compatible with OPCRar, the 5'end of the forward primer EU523377-F-57 was biotinylated in several reactions. The OPCRar solution was heated at 85° C. for 2 minutes to denature the mold, followed by 40 cycles of reciprocating between 15 seconds at 80°C and 15 seconds at 65°C. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. A 213 bp product was clearly observed in all samples, consistent with the expected length of the OPCRar target sequence (FIG. 13 ).

* [00 128] Referring now to Figure 13, there is shown the gel of the specific target sequence is present in the bacterial genomic DNA amplification in accordance with an embodiment of the invention. Total nucleic acid (1.1 ng/μL) isolated from C. Liberibacter asiaticus-infected leaf tissue was used as a starting template. All reactions were performed in the presence of 15% DMSO, using VentR(exo-) DNA polymerase, Et SSB, and primers EU523377-F-57 and EU523377-R-56. Primer EU523377-F-57 was biotinylated or unmodified at the 5'end of the oligonucleotide. The OPCRar solution was heated at 85[deg.] C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 15 seconds at 80[deg.] C. and 15 seconds at 65[deg.] C. were performed. The amplified product was visualized on a 12% acrylamide gel stained with ethidium bromide. For all positive samples, 213 bp products were clearly observed, consistent with the expected length of the OPCRar target sequence, but not in the negative samples.

Example 6: organelles DNA Amplification method of specific sequence on phase

[00129] In order to describe that OPCRar has the ability to amplify specific target sequences present in organelle DNA, in the case of chloroplast DNA, the present inventors used the OPCRar primer pair, rbcL_For and rbcL_Rev, C. riberi. A 137 bp fragment of the plant rbcL gene was generated from total nucleic acids isolated from plant tissues infected with or not infected with Liberibacter asiaticus. OPCRar buffer (10X) was prepared in advance and contained 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. 20 μL of OPCRar solution was prepared by combining the following:

8.4 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets rbcL_For and rbcL_Rev (2 or 4 μM respectively)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single stranded binding protein (500 μg/mL)

Total nucleic acid isolated from 2.0 μL leaf tissue (3.3 ng/μL)

In order to determine the threshold of the primer concentration required to effectively amplify the rbcL gene fragment, two different concentrations of primer pairs rbcL_For and rbcL_Rev were used. The reaction was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 10 seconds at 76° C. and 10 seconds at 60° C. were performed. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. A 137 bp product was clearly observed in both the infected and uninfected samples, which was consistent with the expected length of the OPCRar target sequence (FIG. 14 ).

Referring now to Figure 14, the amplification of a specific target sequence present in the chloroplast DNA according to an embodiment of the present invention exists in the gel. Total nucleic acids (3.3 ng/μL) isolated from C. Liberibacter asiaticus-infected ( i ) or non- infected (ii) leaf tissue were used as a starting template. All reactions were performed using VentR (exo-) DNA polymerase, Et SSB and primers rbcL_For and rbcL_Rev in the presence of 10% DMSO. The OPCRar solution was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 76° C. for 10 seconds and 60° C. for 10 seconds were performed. The amplified product was visualized on a 12% acrylamide gel stained with ethidium bromide. K A 137 bp product was clearly observed for all positive samples, which was consistent with the expected length of the OPCRar target sequence.

Example 7: OPCRar Complex amplification method of target sequence and positive control by

[00132] In order to describe that OPCRar can amplify a number of specific target sequences, the present inventors use OPCRar primer pairs hyvl_For/hyvl_Rev and rbcL_For / rbcL_Rev from plant tissues infected with C. reveribacter asiaticus. The extracted nucleic acid was amplified. These primer sets generate 139 and 137 bp products, respectively. OPCRar buffer (10X) was prepared in advance and contained 400 mM Tris-HCl (pH 8.4), 10 mM ammonium sulfate, 100 mM potassium chloride and 0.25% Triton X-100. A 20 μL OPCRar solution was prepared by combining the following:

6.4 μL water

2.0 μL 10x OPCRar buffer

3.0 μL DMSO

0.4 μL potassium chloride (2 M)

0.5 μL magnesium chloride (100 mM)

0.5 μL dithiothreitol (100 mM)

0.5 μL dNTPs (10 mM)

2.0 μL primer sets rbcL_For and rbcL_Rev (2 or 3 μM, respectively)

2.0 μL primer sets hyvl_For and hyvl_Rev (8 μM each)

0.5 μL VentR (exo-) DNA polymerase (2 U/μL)

0.2 μL Et SSB, extremely thermostable single stranded binding protein (500 μg/mL)

Total nucleic acids isolated from 2.0 μL leaf tissue (3.3 ng/μL)

[00133] hyvl In the presence of 800 nM primers specific for the gene fragment, two different concentrations of primer pairs rbcL_For and rbcL_Rev were used to determine the threshold of the primer concentration required to efficiently amplify the rbcL gene fragment. The reaction was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 10 seconds at 76° C. and 10 seconds at 60° C. were performed. After the reaction was complete, 5 μL of the OPCRar product was mixed with 2 μL of 6X sample loading buffer (New England BioLabs) and 1 μL of formamide, hung on a 12% acrylamide gel, and visualized with ethidium bromide. Consistent with the expected length of the OPCRar target sequence, both 139 bp and 137 bp products were clearly observed. For clarity, the OPCRar product generated using two primer pairs alone (Examples 3 and 6) was walked in parallel with the complex reaction (FIG. 15).

[00134] Referring now to FIG. 15, there is a gel representing a complex amplification product of two target sequences according to an embodiment of the present invention. Total nucleic acid (3.3 ng/μL) isolated from C. Reberrybacter asiaticus-infected leaf tissue was used as a starting template. All reactions were performed in the presence of 10% DMSO, using VentR (exo-) DNA polymerase, Et SSB and primer sets hyvl_For/hyvl_Rev and rbcL_For/rbcL_Rev at the indicated concentrations. The composite OPCRar solution was heated at 85° C. for 2 minutes to denature the mold, and then 40 cycles of reciprocating between 10 seconds at 76° C. and 10 seconds at 60° C. were performed. The composite product was visualized on a 12% acrylamide gel stained with ethidium bromide. When compared to the OPCRar product generated from each primer set alone (see FIGS. 11 and 15), both 139 and 137 bp products were clearly observed.

[00 135] With reference now to Figure 16, there is an amplification product derived from the presence of Et reaction SSB shown in accordance with one embodiment of the invention. SSB improves amplification efficiency at low melting points using the system and method of the present invention. The present inventors studied the use of "ET SSB", an extremely thermostable single-stranded binding protein, in order to show that it aids in the performance of OPCRar at a specific melting point. The OPCRar reaction was performed against HLB pathogens using purified leaf sample DNA containing the HLB disease gene. The template was DNA purified from citrus leaves of C. reberrybacter infected trees. Primers were hyvl_For/ and hyvl_Rev. The thermocycler conditions were as follows: 40 cycles of initial melting at 85°C for 2 minutes, then denaturing at 76°C or 74°C for 10 seconds and annealing at 60°C for 10 seconds. Results indicated in the presence of Et SSB resulted in amplification of the C. Liberibacter target in all analyzed temperature regimens. In the presence of ET SSB protein and in the absence of ET SSB, comparative OPCRar experiments were performed twice. A standard PCR thermocycler with precise temperature control was used to evaluate the amplification performance with and without SSB. The results indicated that we harvested HLB amplified at 74° C. in the presence of ET SSB, but was not observed without ET SSB.

[00136] Referring now to FIG. 17, gel electrophoresis of an OPCRar reaction performed using hyvl_For and hyvl_Rev primers and purified C. reveribacter DNA isolated from the leaves of infected trees according to an embodiment of the present invention is performed. Wherein the reaction is one that does not include the ramping parameters associated with a typical PCR thermocycler FIG. 17A or includes a ramping time for the cycling temperature FIG. 17B. As used herein, ramping means heating.For example, in each thermal cycling, the heating process of increasing the amplification reaction temperature from the annealing temperature to the denaturation temperature is called ramping up, or cooling from the denaturation temperature to the annealing temperature is called ramp down. . All traditional PCR cyclers and methods have a controlled heating design, where ramping up is about >3 degrees/sec and ramping down rate about >1 degrees/sec. The ramping time is not included in the typical cycling profile. During the denaturation, annealing and stretching steps, the instrument does not start counting time until the desired temperature is reached. For example, if the denaturation time is 90 to 10 seconds, the instrument will not start counting 10 seconds until 90 degrees is reached. In contrast, the system and method of the present invention is a low-cost heater designed to mean that, for example, 10 seconds at 80°C means that when the heating process begins (rather than waiting for the desired denaturation temperature to be reached), time counting begins. Provide the device. Figure 17 A: The inventors of the low cost, thermal heater engine (no active cooling and precise temperature control,> + 2 degree fluctuations, (see e.g., systems and devices disclosed in 61/477,357) among HLB diseases (Citrus Green Disease Pathogens). , Huang Long Bing) OPCRar amplification of the target was tested.

In a device developed by Mesa Tech International, Inc. (MTI device) or a traditional PCR thermocycler (PCR thermocycler), OPCRar 20 μL reaction was performed in a micro-heater reaction chamber under a microprocessor. , As soon as the command to change the temperature was executed by the microprocessor, a holding time of 10 seconds was calculated for each temperature part of the program when calculated (ie, no ramping time FIG. 17A). This is the opposite of the 10 second holding time calculated for each temperature portion of the program immediately after the target temperature is detected by the temperature sensor located in the reaction well (ie, ramping time Fig. 17B). The PCR thermocycler conditions were as follows: initial melting at 85° C. for 2 minutes, then 40 cycles of 80° C. for 10 seconds and 60° C. for 10 seconds. The conditions of the MTI apparatus were as follows: initial melting at 85°C for 2 minutes, 40 cycles of 10 seconds at 82°C and 20 seconds at 59°C.

[00 137] Figure 17A: the first lane, and a separate amplifier without OPCRar ramp (20 μL reaction). From left: 50 bp standard DNA size strand; Lanes 2 and 3: OPCRar amplification reactions (2x, 2nd and 3rd lanes) in a standard PCR thermocycler with ramp step and precise temperature control. Initial melting: 40 cycles repeated between 2 minutes at 85° C., denaturation at 80° C. for 10 seconds and annealing at 60° C. for 10 seconds. Lanes 4 and 5: OPCRar amplification performed in low cost heat engines without active cooling and/or separate ramping adjustments. The thermal engine ramps down to 80° C. denaturation or 60° C. annealing temperature from ambient field temperature (~ 25° C.) compared to having no ramping step for denaturing or annealing temperature. HLB OPCRar was performed using purified plant DNAFMF containing the HLB disease target sequence, in a 20 μL reaction. The present inventors used a PCR thermocycler for a positive control for the MTI device test. The PCR thermocycler conditions were as follows: MTI apparatus for no ramping The thermocycler conditions were as follows: cycling between initial melting at 85° C. for 2 minutes, denaturation at 82° C. for 10 seconds and annealing at 59° C. for 20 seconds. Cycling was repeated 40 times. The MTI device thermocycler for the ramping up phase was the same condition except there was a ramp up step of <10 seconds and a ramp down phase of <20 seconds. The data presented by HLB OPCRar was able to still amplify the HLB amplicon, even though there were no ramp up and ramp down steps. Comparison of 17A and 17B showed a significant improvement in amplification yield, given the ramp time.

[00138] Now, referring to FIG. 18, in the thermal register-based low-cost amplification device of MIT, two primer sets as described above: 1) C. Primers hyvl_For and hyvl_Rev specific to C. Reveriebacter DNA targets and 2) Citrus housekeeping Gene rbcL . Complex OPCRar reactions according to an embodiment of the present invention including primers rbcl_ (ribulose-1,5-biphosphate carboxylase oxygenase) For and rbcl_Rev specific to the amplification product are shown in the gel. The reaction products from the PCR amplification described and performed in a low cost heater with no ramping (e.g., 5 degrees/sec in a PCR apparatus) and without precise temperature control were hung on the gel. In a PCR thermocycler and MTI device (with or without ramping program), we tested the RbCL internal positive control with HLB primer. The present inventors performed a 40 uL reaction with a purified HLB sample. PCR normal cycler conditions were as follows: Initial melting: cycling between 85° C. for 2 minutes, 80° C. for 10 seconds denaturation and 60° C. for 10 seconds annealing. MTI apparatus amplification conditions were as follows: initial melting: cycling between 90°C for 2 minutes, 82°C for 10 seconds denaturation and 59°C for 20 seconds annealing. We have found that it is still possible to amplify both HLB and RbCL primer sequences without ramping. The HLB product is near -147 bp, and the RbCL product is near -140 bp. The MTI device was comparable to the PCR thermocycler product for both conditions. The forward primer of this reaction is ccagccttga tcgttacaaa gggcgatgct acaacatt (SEQ ID NO 9) (Tm is about 73.9C (10% DMSO, 76/60C), and the reverse primer is iscatgttagta acagaacctt cttcaaaaag gtctaacggg taa (SEQ ID NO 10) (Tm is About 71.2 C (10% DMSO, 76/60 C) The OPCRar reaction was performed with or without ramp time in temperature hold times (as described in Figures 16 and 17) as indicated. A 40 μL reaction was carried out using purified citrus leaf DNA derived from C. Reveriebacter, PCR thermocycler control conditions were as follows: Initial melting: 85°C for 2 minutes, 80°C for 10 seconds and 60°C 40 cycles of 10 seconds at 40. MTI device amplification conditions were as follows: Initial melting: 40 cycles of 2 minutes at 90° C., 10 seconds at 82° C. and 20 seconds at 59° C. The results do not include the ramp time in the holding time calculation. Without, it has been shown that the MTI device is capable of amplifying both C. reveribacter and rbcL sequences, the C. reveribacter product is approximately 147 bp (HLB) and the rbcL (RbCL) product is approximately 140 bp.

[00139] The melting point (Tm) of all primers was calculated using IDT oligo analyzer 3.1 (Integrated DNA Technologies, Inc., Coralville, IA) using primer 3 Tm calculation software, where salt, dNTP, Mg, primer The concentration parameter is considered using the following parameters:

Oligonucleotide concentration: 0.25 μM; Na ⁺ concentration: 50mM; Mg ⁺⁺ concentration = 2.5 mM; dNTPs concentration = 0.25 μM. The symbol "a" means adenine, the symbol "g" is guanine, "c" is cytosine, "t" is thymine, "u" is uracil, "r" is purine, "y" is pyrimidine, "m" is amino, "k" is keto, "n" means a or g or c or t/u, unknown or any other.

[00140] (SEQ ID NO 1)

Access Number : CY087034

Form : viral RNA

Length : 1010

Organism : Influenza A virus (H1N1)

Other information : matrix protein 2 (M2) and matrix protein 1 (M1) genes

[00141] (SEQ ID NO 2)

Form : Forward primer

Name : FP3

Length : 46

Tm: average 75 ℃

[00142] (SEQ ID NO 3)

Form: Reverse primer

Name: RP3

Length: 40

Tm: average 77.8 ℃

[00143] (SEQ ID NO 4)

Form: Reverse primer

Name: RP4

Length: 46

Tm: average 74.7 ℃

[00144] (SEQ ID NO 5)

Form: Forward Primer

Name: UniAfCDC

Length: 22

Tm: average 65.0 ℃

[00145] (SEQ ID NO 6)

Form: Reverse primer

Name: UniArCDC

Length: 24

Tm: average 66.6 ℃

[00146] (SEQ ID NO 7) FAM-tgcagtcctc gctcactggg cacg-BHQ

Form: TaqMan probe

Name: UniApCDC

Length: 24

Tm: 73.4 ℃

[00147] (SEQ ID NO 8)

Access Number : AB505957

Form: Chloroplast DNA

Length: 1326

Organism: Citrus (Citrus sinensis)

Other information: rbcL , ribulose-1,5-biphosphate carboxylase/oxygenase laage subunit

[00148] (SEQ ID NO 9)

Form: Forward Primer

Name: rbcL_For

Length: 38

Tm: 73.9 ℃

[00149] (SEQ ID NO 10)

Form: Reverse primer

Name: rbcL_Rev

Length: 43

Tm: 71.2 ℃

[00150] (SEQ ID NO 11)

Accession number: from EU523377

Form: bacterial DNA

Length: 890

Organism: Candidatus reberrybacter asiaticus (Candidatus Liberibacter asiaticus)

Other information: elongation factor Ts

[00151] (SEQ ID NO 12)

Form: Forward Primer

Name: NBEU523377-F-57

Length: 57

Tm: 75.8 ℃

[00152] (SEQ ID NO 13) [Biotin-5]tcttcgtatc ttcatgcttc tccttctgag ggtttaggat cgattggtgt tcttgta

Form: Biotinylated Forward Primer

Name: EU523377-F-57

Length: 57

Tm: 75.8 ℃

[00153] (SEQ ID NO 14)

Form: Forward Primer

Name: HLBForSh

Length: 47

Tm: 75.6 ℃

[00154] (SEQ ID NO 15)

Form: Reverse primer

Name: EU523377-R-56

Length: 56

Tm: 75.8 ℃

[00155] (SEQ ID NO 16)

Form: Reverse primer

Name: HLBRevSh

Length: 49

Tm: 75.5 ℃

[00156] (SEQ ID NO 17)

TARGET: Candidatus reberrybacter asiaticus 16S ribosomal RNA

Form: forward primer (underlined), including 5'detection sequence

Name: HLBas-P2

Length: 39

Tm: 62.7 ℃

[00157] (SEQ ID NO 18)

TARGET: Candidatus reberrybacter asiaticus 16S ribosomal RNA

Form: reverse primer (underlined), with 5'T7 promoter

Name: HLBr-P1

Length: 56

Tm: 64.5 ℃

[00158] (SEQ ID NO 19)

Form: Forward Primer

Name: hyvl_For

Length: 45

Tm: 72.2 ℃

[00159] (SEQ ID NO 20)

Form: Reverse primer

Name: hyvl_Rev

Length: 51

Tm: 70.9 ℃

[00160] Even if the invention has been described in detail with specific references to the described embodiments, the same results may be obtained for other embodiments. Changes and modifications of the present invention will be apparent to those skilled in the art, and are intended to cover all such modifications and equivalents. The entire disclosure of all patents and publications cited above is incorporated herein by reference.

SEQUENCE LISTING <110> Mesa Tech International, Inc. <120> Oscillating Amplification Reaction for Nucleic Acids <130> 33106-PCT2 <140> 61/477,357 <141> 2011-04-20 <150> 61/477,437 <151> 2011-04-20 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 1010 <212> DNA <213> Influenza A virus <220> <221> misc_feature <222> (993)..(993) <223> n is a, c, g, or t <220> <221> misc_feature <222> (1002)..(1002) <223> n is a, c, g, or t <400> 1 taaagatgag tcttctaacc gaggtcgaaa cgtacgttct ttctatcatc ccgtcaggcc 60 ccctcaaagc cgagatcgcg cagagactgg aaagtgtctt tgcaggaaag aacacagatc 120 ttgaggctct catggaatgg ctaaagacaa gaccaatctt gtcacctctg actaagggaa 180 ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg aggactgcag cgtagacgct 240 ttgtccaaaa tgccctaaat gggaatgggg acccgaacaa catggataga gcagttaaac 300 tatacaagaa gctcaaagga gaaataacgt tccatggggc caaggaggtg tcactaagct 360 ttcaactggt gcacttgcca gttgcatggg cctcatatac aacaggatgg gaacagtgac 420 cacagaagct gcttttggtc tagtgtgtgc cacttgtgaa cagattgctg attcacagca 480 tcggtctcac agacaaatgg ctactaccac caatccacta atcaggcatg aaaacagaat 540 ggtgctggct agcactacgg caaaggctat ggaacagatg gctggatcga gtgaacaggc 600 agcagaggcc atggaggttg ctaatcagac taggcagatg gtacatgcaa tgagaactat 660 tgggactcat cctagctcca gtgctggtct gaaagatgac cttcttgaaa atttgcaggc 720 ctaccagaag cgaatgggag tgcagatgca gcgattcaag tgatcctctc gtcattgcag 780 caaatatcat tgggatcttg cacctgatat tgtggattac tgatcgtctt tttttcaaat 840 gtatttatcg tcgctttaaa tacggtttga aaagagggcc ttctacggaa ggagtgcctg 900 agtccatgag ggaagaatat caacaggaac agcagagtgc tgtggatgtt gacgatggtc 960 attttgtcaa catagagcta gagtaataga cgntttgtcc anaatgccct 1010 <210> 2 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> forward primer designed for SEQ ID NO 1 <400> 2 ywctcatgga rtggctaaag acaagaccra tcctgtcacc tctgac 46 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> reverse primer designed for SEQ ID NO 1 <400> 3 agggcattyt ggacaaakcg tctacgytgc agtccycgyt 40 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for SEQ ID NO 1 <400> 4 tttggrtctc cattyccatt tagggcatty tggacaaakc gtctat 46 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer UniAfCDC for SEQ ID NO 1 <400> 5 gaccratcct gtcacctctg ac 22 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer UniArCDC designed for SEQ ID NO 1 <400> 6 agggcattyt ggacaaakcg tcta 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TAQman probe UniApCDC <400> 7 tgcagtcctc gctcactggg cacg 24 <210> 8 <211> 1326 <212> DNA <213> Citrus sinensis <400> 8 tgttggattc aaggccggtg ttaaagatta taaattgact tattatactc ctgactatgt 60 aaccaaagat actgatatct tggcagcatt ccgagtaact cctcagcccg gagttccacc 120 cgaggaagcg ggggctgcgg tagctgcgga atcttctact ggtacctgga cagctgtgtg 180 gaccgatggg cttaccagcc ttgatcgtta caaagggcga tgctacaaca ttgagcccgt 240 tgctggagaa gagaatcaat atatatgtta tgtagcttac ccgttagacc tttttgaaga 300 aggttctgtt actaacatgt ttacttccat tgtgggtaat gtatttggtt tcaaagcact 360 gcgcgctcta cgtctagagg atctacgaat ccctcctgcg tatactaaaa ctttccaagg 420 cccgcctcac ggcatccaag ttgagagaga taaattgaac aagtatggcc gtcccctgtt 480 gggatgtact attaaaccta aactggggtt atccgcgaag aattatggta gggcggttta 540 tgaatgtcta cgtggtggac ttgactttac caaagatgat gagaacgtga actcccaacc 600 atttatgcgt tggagggacc gtttcttatt ttgtgcggaa gctctttata aagcgcaagc 660 tgaaacaggt gaaatcaaag gtcattactt gaatgctact gcagggacat gcgaagaaat 720 gctaaaaagg gctgtctttg ccagagagtt gggagttcct atcgtaatgc atgactactt 780 aacaggggga ttcaccgcaa atactacctt ggctcattat tgccgagata atggtctact 840 tcttcacatc caccgtgcaa tgcatgcagt tattgataga cagaagaatc atggtatgca 900 ctttcgtgta ctagctaaag ctttgcgtct gtctggtgga gatcatattc acgccggtac 960 agtagtaggt aaacttgagg gggaaagaga cataaccttg ggatttgttg atttactacg 1020 tgatgatttt gttgaaaaag atcgaagccg cggtatttat ttcactcaag attgggtctc 1080 tataccaggt gttatacctg tggcttccgg gggtattcac gtttggcata tgcctgcgtt 1140 gacagagatc tttggagatg attccgtatt acaatttggt ggaggaactt taggacaccc 1200 ttggggaaat gcacccggcg ctgtagctaa tcgagtagct ctagaagcat gtgtacaagc 1260 tcgtaatgaa ggacgcgatc ttgctcgtga aggtaatgaa attatccggg aggctagcaa 1320 atggag 1326 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer rbcL_For <400> 9 ccagccttga tcgttacaaa gggcgatgct acaacatt 38 <210> 10 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> reverse primer rbcL_Rev <400> 10 catgttagta acagaacctt cttcaaaaag gtctaacggg taa 43 <210> 11 <211> 890 <212> DNA <213> Candidatus Liberibacter asiaticus <400> 11 atgagtaagg tatctgctgt tgcggtaaag gagttacgtg ggaaaactgg tgcaggtatc 60 ttggactgca agaatgccct tttggaggct aagggtgata gtgaattagc gattgatatt 120 ttgcgcacta aaggagctat ggcagctagt aagagagagg gtagaaaggt ttcagagggg 180 cttataggaa ttgctcgtga tggatataag aaggcatcga ttgtagaagt caatgttgag 240 accgatagtt tggcaaaaaa cactgatttt cagagtcttg tttctaatat tgcgggtatt 300 gctctttcca ccgatggttc tttggacaat gttcttgcga tgccatttga ccatagtggg 360 attactgtgg gggatggaat taagcagcag attgctatca ccggtgaatg tattaagctg 420 aggcgttccg ctcttctgtg tgtttcggaa ggggtcatct cttcgtatct tcatgcttct 480 ccttctgagg gtttaggatc gattggtgtt cttgtagcgt tgcagtcttc tgcggaagat 540 aaggaattgc tttctgcgat tggagagaag attgcagtgc atgtaatgct ggcttctcct 600 tcggtgattt ctgtgcagat gcttgatcct tctattgttg ctaataaacg tgcccattac 660 atgacagaag cacttgattc tgggaaatca ggtaatattg ttgaaaagat cgtaaatgga 720 aagatgcaaa gcttttgtaa ggaatgtgtt cttttgcatc aggggtttgt tgttgatcct 780 tccaaaactg tatcagattt tttgaaagaa tctgaaaaat caatcggtgc ttctattgaa 840 gttgttggtg tatctcattt tgttgtgggt aaagaaaatg atgatggtta 890 <210> 12 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> forward primer NBEU523377-F-57 <400> 12 tcttcgtatc ttcatgcttc tccttctgag ggtttaggat cgattggtgt tcttgta 57 <210> 13 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> biotinylated forward primer EU523377-F-57 <400> 13 tcttcgtatc ttcatgcttc tccttctgag ggtttaggat cgattggtgt tcttgta 57 <210> 14 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> forward primer HLBForSh <400> 14 cgattggtgt tcttgtagcg ttgcagtctt ctgcggaaga taaggaa 47 <210> 15 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> reverse primer EU523377-R-56 <400> 15 tgcttctgtc atgtaatggg cacgtttatt agcaacaata gaaggatcaa gcatct 56 <210> 16 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> reverse primer HLBRevSh <400> 16 aacaatagaa ggatcaagca tctgcacaga aatcaccgaa ggagaagcc 49 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer (underlined), contains 5'detection sequence HLBas-P2 <400> 17 gatgcaaggt cgcatatgag gagcgcgtat gcaatacga 39 <210> 18 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (underlined), contains 5'T7 promoter HLBr-P1 <400> 18 aattctaata cgactcacta tagggagaag ggcgttatcc cgtagaaaaa ggtaga 56 <210> 19 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> forward primer hyvl_For <400> 19 ggccgtttta acacaaaaga tgaatatcat agatggggta gtcaa 45 <210> 20 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> reverse primer hyvl_Rev <400> 20 cggccatttt agataaatca atttgttcta gtttagatac atcaatttgt t 51 <210> 21 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 21 gttcttgtag cgttgcagtc ttctgcggaa gataaggaat tgcttt 46 <210> 22 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 22 gggcacgttt attagcaaca atagaaggat caagcatctg cacagaaat 49 <210> 23 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 cttgtagcgt tgcagtcttc tgcggaagat aaggaattgc tttctgcg 48 <210> 24 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 cacgtttatt agcaacaata gaaggatcaa gcatctgcac agaaatcacc g 51 <210> 25 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 ggtgttcttg tatcgttgca gtcttctgcg gaagataagg aattgcttt 49 <210> 26 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 gtaatgggca cgtttattag caacgataga aggatcaagc aactgcacag aaat 54 <210> 27 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 27 cttgtatcgt tgcagtcttc tgcggaagat aaggaattgc tttctgcg 48 <210> 28 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 28 ggcacgttta ttagcaacga tagaaggatc aagcatctgc acagaaatca ccg 53

Claims (33)

A method for selectively amplifying a template of a target nucleic acid sequence contained in a sample,
A primer complementary to the template of the target nucleic acid sequence and
Contacting the sample with an amplification reaction mixture containing 8-15 vol% concentration of nucleic acid destabilizing agent comprising DMSO, formamide or betaine;
Reciprocating the reaction temperature between an upper limit denaturation temperature and a lower limit annealing temperature with a temperature difference of up to 20 DEG C during several temperature cycles; And
Amplifying the template of the target nucleic acid sequence
&Lt; / RTI &gt; The method of claim 1, wherein the temperature difference during a number of temperature cycles is at most 15 ° C. The method of claim 1, wherein the temperature difference during a number of temperature cycles is at most 10 ° C. The method of claim 1, wherein the temperature difference during a number of temperature cycles is at most 5 ° C. The method of claim 1, wherein after reaching the upper or lower temperature, the temperature is maintained for a predetermined time within the temperature variation. The method according to claim 1, wherein the temperature changes to another temperature within the temperature range after reaching the upper limit temperature or the lower limit temperature. The method of claim 1 wherein the lower limit temperature is at least 50 ° C. The method of claim 1, wherein the upper limit temperature is at most 85 ° C. 2. The method of claim 1, wherein the template of the target nucleic acid sequence is single stranded DNA or RNA. 2. The method of claim 1, wherein the template of the target nucleic acid sequence is double stranded DNA or RNA. 2. The method of claim 1, wherein the template of the target nucleic acid sequence is RNA. 2. The method of claim 1, wherein the template of the target nucleic acid sequence is DNA. 2. The method of claim 1, wherein the length of the target nucleic acid is less than 1000 bp. 2. The method of claim 1 wherein the length of the target nucleic acid is less than 250 bp. 2. The method of claim 1, wherein the length of the target nucleic acid is less than 150 bp. 2. The method of claim 1, wherein the length of the target nucleic acid is less than 100 bp. A method for amplifying a template of a target nucleic acid sequence contained in a sample,
A primer or primer pair comprising a length of 35 to 70 base pairs and complementary to a template of the target nucleic acid sequence wherein the melting point of each primer of the pair of primers is from 70 to 80 DEG C,
DMSO at a concentration of 8-15 vol%
Monovalent cation,
Divalent cation,
dNTP, and
DNA polymerase
Contacting the sample with the amplification reaction mixture containing the amplification reaction mixture;
Reciprocating the reaction temperature between an upper limit denaturation temperature and a lower limit annealing temperature with a temperature difference of up to 20 DEG C during several temperature cycles; And
Amplifying the template of the target nucleic acid sequence
&Lt; / RTI &gt; 18. The method of claim 17, wherein the divalent cation is a salt selected from the group consisting of magnesium, manganese, copper, zinc, or combinations thereof. 18. The method of claim 17, wherein the monovalent cation is a salt selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium, or combinations thereof. 2. The method of claim 1, wherein the amplification reaction comprises a DNA polymerase. 21. The method of claim 17 or 20, wherein the DNA polymerase is a thermostable DNA polymerase. 21. The method of claim 17 or 20, wherein the DNA polymerase is selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase and DeepVentR DNA polymerase. 21. The method according to claim 17 or 20, wherein the DNA polymerase has an activity to replace the strand. 21. The method of claim 17 or 20, wherein the DNA polymerase does not have 3 '-> 5' exonuclease activity. 12. The method of claim 11, wherein the amplification reaction mixture comprises a reverse transcriptase and a DNA polymerase. 26. The method of claim 25, wherein the reverse transcriptase is a thermostable reverse transcriptase. 26. The method of claim 25, wherein the reverse transcriptase is selected from AMV-RT, Superscript II reverse transcriptase, SuperScript III reverse transcriptase, or MMLV-RT. 18. The method according to any one of claims 1 to 17, wherein the amplification reaction mixture contains a single strand binding protein (SSB protein). 29. The method of claim 28, wherein the single strand binding protein is a thermostable single strand binding protein. 29. The method of claim 28, wherein the single strand binding protein is a thermostable single strand binding protein. 18. The method according to any one of claims 1 to 17, wherein the sample is not alcohol free. 18. The method according to any one of claims 1 to 17, wherein the sample is not salt free. The method of claim 1, wherein the amplification reaction mixture comprises an amplification reaction mixture buffer comprising:
Single- or double-stranded nucleic acid destabilizing agents;
Monovalent cations;
Divalent cation;
dNTP; And
DNA polymerase.

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